Received July 24, 2007; Revision received August 20, 2007
Interactions of APE1 (human apurinic/apyrimidinic endonuclease 1) and
DNA polymerase beta with various DNA structures imitating
intermediates of DNA repair and replication were investigated by gel
retardation and photoaffinity labeling. Photoaffinity labeling of APE1
and DNA polymerase beta was accomplished by DNA containing
photoreactive group at the 3´-end in mouse embryonic fibroblast
(MEF) cell extract or for purified proteins. On the whole, modification
efficiency was the same for MEF-extract proteins and for purified APE1
and DNA polymerase beta depending on the nature of the
5´-group of a nick/gap in the DNA substrate. Some of DNA duplexes
used in this work can be considered as short-patch (DNA with the
5´-phosphate group in the nick/gap) or long-patch (DNA containing
5´-sugar phosphate or 5´-flap) base excision repair (BER)
intermediates. Other DNA duplexes (3´-recessed DNA and DNA with
the 5´-hydroxyl group in the nick/gap) have no relation to
intermediates forming in the course of BER. As shown by both methods,
APE1 binds with the highest efficiency to DNA substrate containing
5´-sugar phosphate group in the nick/gap, whereas DNA polymerase
beta binds to DNA duplex with a mononucleotide gap flanked by
the 5´-p group. When APE1 and DNA polymerase beta are both
present, a ternary complex APE1-DNA polymerase beta-DNA is
formed with the highest efficiency with DNA product of APE1
endonuclease activity and with DNA containing 5´-flap or
mononucleotide-gapped DNA with 5´-p group. It was found that APE1
stimulates DNA synthesis catalyzed by DNA polymerase beta, and a
human X-ray repair cross-complementing group 1 protein (XRCC1)
stimulates APE1 3´-5´ exonuclease activity on
3´-recessed DNA duplex.
KEY WORDS: APE1, base excision repair, photoaffinity labeling